Purpose Accumulating evidence shows that effort–reward imbalance (ERI) at work can cause various health problems. However, few studies have investigated the biological pathways linking ERI and health outcomes, and their findings have been inconsistent. In this study, we investigated the associations between ERI, the hypothalamic–pituitary–adrenocortical axis, and inflammation in a sample of police officers. Methods One hundred forty-two male police officers that were engaged in a working system of 24-h shifts were followed up during the work shift as well as during the two subsequent work-free days. Throughout this period, the participants provided two saliva samples each day for the 3-day period, and we measured the concentrations of cortisol and C-reactive protein (CRP) in the saliva. The police officers also completed the Japanese short version of the Effort–Reward Imbalance Questionnaire. Results The results of linear mixed model analyses controlled for possible confounding variables indicated that higher effort scores ( p  = 0.031) as well as effort–reward ratio ( p  = 0.080) were associated with lower cortisol levels, and the effect of effort was strengthened in the younger police officers ( p  = 0.017). Furthermore, higher effort scores were associated with higher CRP levels in younger police officers ( p  = 0.037). Conclusions Our results indicate that effort, a component of ERI, has physiological effects in younger police officers, which possibly contribute to the development of stress-related diseases.